5-hydroxybenzimidazole and 5-6-dimethylbenzimidazole

Organisms that perform the de novo biosynthesis of cobalamin (vitamin B12) do so via unique pathways depending on the presence of oxygen in the environment. The anaerobic biosynthesis pathway of 5,6-dimethylbenzimidazole, the so-called "lower ligand" to the cobalt center, has been recently identified. This process begins with the conversion of 5-aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (HBI) by the radical S-adenosyl-l-methionine (SAM) enzyme BzaF, also known as HBI synthase. In this work we report the characterization of a radical intermediate in the reaction of BzaF using electron paramagnetic resonance spectroscopy. Using various isotopologues of AIR, we extracted hyperfine parameters for a number of nuclei, allowing us to propose plausible chemical compositions and structures for this intermediate. Specifically, we find that an aminoimidazole radical is formed in close proximity to a fragment of the ribose ring. These findings induce the revision of past proposed mechanisms and illustrate the ability of radical SAM enzymes to tightly control the radical chemistry that they engender.

Topics: Anaerobiosis; Bacterial Proteins; Benzimidazoles; Biosynthetic Pathways; Desulfuromonas; Electron Spin Resonance Spectroscopy; S-Adenosylmethionine; Vitamin B 12

The transformation of [1-15N]5-hydroxybenzimidazole and [1-15N]5-hydroxy-6-methylbenzimidazole into the 5,6-dimethylbenzimidazole moiety of vitamin B12 by Eubacterium limosum-cultures was studied. The vitamin B12 obtained was exclusively 15N-labeled in N-1 of the base part, as revealed by NMR-measurements. This indicates that either the unsubstituted 5,6-dimethylbenzimidazole presumably formed is not released from the enzyme until the ribose-5'-phosphate substituent is introduced, or that the precursors are first transformed into their alpha-nucleotide-5'-phosphates which then react to form 5,6-dimethylbenzimidazole-alpha-D-ribofuranoside- 5'-phosphate (alpha-ribazole-5'-phosphate).

Topics: Benzimidazoles; Cobamides; Eubacterium; Magnetic Resonance Spectroscopy; Vitamin B 12

Eubacterium limosum transformed [2-13C]5-hydroxybenzimidazole not only into [2-13C]5-hydroxybenzimidazolylcobamide, but also into [2-13C]5-methoxy-6-methylbenzimidazolylcobamide and into [2-13C]5,6-dimethylbenzimidazolylcobamide (vitamin B12). [2-13C]5-Hydroxy-6-methyl-benzimidazole was used by this bacterium to form [2-13C]5-hydroxy-6-methylbenzimidazolyl-cobamide, [2-13C]5-methoxy-6-methylbenzimidazolylcobamide and [2-13C]5,6-dimethylbenzimidazolylcobamide. The 1H-NMR spectrum of the 5,6-dimethylbenzimidazole isolated from the 13C-labeled vitamin B12 preparations revealed that the externally added bases had been transformed into the vitamin B12 base almost without dilution of the label. This suggests that 5-hydroxybenzimidazole and 5-hydroxy-6-methylbenzimidazole are precursors of 5,6-dimethylbenzimidazole. On the basis of these results, a hypothetical scheme for the biosynthesis of 5,6-dimethylbenzimidazole via 5-hydroxybenzimidazole and 5-hydroxy-6-methylbenzimidazole is discussed. This scheme can also explain the formation of the other benzimidazole bases found in natural vitamin B12 analogs.

Topics: Benzimidazoles; Biotransformation; Eubacterium; Vitamin B 12